DE69635241T2 - LARGE-SIZED TRANSPLANTS FROM SUBMUCOSAL FABRIC AND METHOD FOR THEIR PRODUCTION - Google Patents

Description

technical Field of the invention

These This invention relates to tissue graft constructs for use in delivery of new growth and healing of damaged or diseased tissue structures are usable. More specifically, the invention is directed to submucosal tissue graft constructs from several strips of submucosal tissue of a warm-blooded vertebrate and directed to processes for their preparation.

technical Background and Summary of the Invention

It is known that compositions with tunica submucosa peeled from both the tunica muscularis and at least the lumenal portion of the tunica mucosa of the gut of a warm-blooded vertebrate are useful as a tissue graft. See, for example, B. US 4,902,508 and US 5,281,422 , The compositions described in these patents are characterized by excellent mechanical properties, including high compliance, high burst pressure, and effective porosity index, and such compositions can be used to advantage as vascular graft constructs and connective tissue replacement applications. When used in such applications, the submucosal graft construct appears to serve as a matrix for regrowing the graft-replaced tissue. Remarkably, even in over 600 species-different submucosal implantations of submucosal tissue, tissue graft rejection has never been demonstrated.

matrices from submucosa for use according to the invention are biodegradable collagen based matrices that are high grade preserved collagens, glycoproteins, proteoglycans and glycosaminoglycans in natural Configuration and concentration included. Submucosal tissue of one warm-blooded Vertebrate is an extracellular Collagen matrix for use according to the invention. Submucosal tissue can be obtained from various sources, for example, from intestinal tissue of animals used for meat production cultured including pigs, cattle and sheep or other warm-blooded vertebrates. Vertebrate submucosal tissue is a copious by-product commercial meat production and therefore cost-effective Tissue graft material.

A restriction the submucosal engraftment constructs described in the above patents is that the Size of the graft by the size of the parent material, from which the submucosal tissue is produced is limited. For example is the size of one from intestinal tissue produced submucosal graft by length and Limited scope of the source intestinal tissue parts. Various Uses of graft constructs from submucosal tissue, including Repair of fractures, Skin grafts, meningeal covers, repair of gastroschisis (congenital abdominal wall defect) and replacement of organ tissue often larger leaves of Graft material as produced directly from natural sources can be.

Large leaves Submucosal tissue can from smaller parts of submucosa tissue by conventional Techniques such as weaving, knitting or the use of adhesives getting produced. The commercial realization of such techniques However, it is often impractical and expensive. In addition, the use of Adhesives or chemical pretreatment to increase the adhesion degrade the biotropic properties of the submucosal graft. Therefore, there is a need after a cheap, easily produced large-area submucosal tissue graft, that retains its biotropic properties.

To an embodiment of the invention become large-area submucosal tissue grafts from several pieces formed by matrices from vertebrate submucosa. Uniform leaves of Submucosal tissue (i.e., one-piece grafts) according to the invention Merging of several strips of submucosal tissue under Forming a tissue sheet having a surface area greater than any of the submucosal tissue sub-strips produced. This process includes the steps of overlapping at least part of a strip of submucosal tissue with at least a part of another strip of submucosal tissue and application by pressure on at least the overlapping ones Parts under conditions involving drainage of the submucosal tissue allow. Under these conditions, the overlapping parts are "fused" and form a uniform one great Tissue sheet. The inventively formed large-area graft consists essentially of submucosal tissue, possibly free of deteriorating adhesives and chemical pretreatment, and have a larger area and higher mechanical strength than that in the formation of the graft used single strips on.

Out Tissue warm-blooded Vertebrates produced single-strip submucosal tissues have directional mechanical properties (i.e., the physical properties are longitudinal different axes different). These directional properties become mainly determined by the orientation of the collagen in the tissue. In intestinal submucosal tissue the collagen fibers are the load-bearing components and these are mainly aligned parallel to the axis of the intestinal lumen. This longitudinal alignment of collagen in intestinal submucosa sagewebe contributes to directional dependence physical properties of submucosal tissue constructs.

To an embodiment of the invention becomes a uniform pseudoisotropic multilayer graft made from many strips of submucosal tissue. The one used here Expression "pseudoisotropic" describes a graft with approximate same physical properties in each axis direction of the Material. The inventive pseudoisotropic Multilayer grafts are made from single strips of submucosal tissue or from leaves of submucosal tissue containing strips of submucosal tissue, produced. The manufacturing process for the pseudoisotropic grafts comprises the overlapping a portion of a first strip (or sheet) with a second Strip (or sheet), with the second strip (or sheet) aligned in a plane parallel to the first strip (or sheet), but turned so that the longitudinal axis of the first strip (or sheet) with the axis of the second strip (or leaf) forms an angle. Other stripes (or leaves) can be found in added in the same way be laminated to a multi-layer structure with the desired number To create layers. The individual strips (or leaves) submucosa are then applied by applying pressure to at least the overlapping ones Parts of submucosal tissue fixed to each other and form a uniform pseudoisotropic multilayer construct.

Short description the drawings

1 Figure 3 is a schematic representation of a homolaminate graft formed from multiple strips of submucosal tissue;

2 Figure 3 is a schematic representation of a pseudoisotropic heterolaminate graft formed from four strips of submucosal tissue;

3 Figure 3 is a schematic representation of a pseudoisotropic heterolaminate graft formed from three sheets of submucosal tissue, each sheet formed from multiple strips of submucosal tissue; and

4 is a schematic representation of a device according to the invention suitable for forming perforations in submucosal tissue grafts.

incoming Description of preferred embodiments

According to the invention is a Method of forming tissue graft constructs with large leaves Submucosal tissue provided. Further, the present invention gives a method for producing pseudoisotropic multilayer sheets Submucosal tissue on. The method includes the step of merging several strips of submucosal tissue forming uniform leaves submucosal tissue.

For the use Submucosal tissue suitable for forming the present graft constructs comprises Naturally linked extracellular Matrix proteins, glycoproteins and other factors. A source of submucosal tissue intestinal tissue is warm-blooded Vertebrates. A preferred source of submucosal tissue for use in this invention is small intestinal tissue.

suitable Intestinal submucosal tissue typically those of both the tunica muscularis and at least tunica submucosa peeled off from the lumenal part of the tunica mucosa. In a inventive embodiment comprises the intestinal submucosal tissue the tunica submucosa and base parts of the Including tunica mucosa the lamina muscularis mucosa and the stratum compactum. These layers are known to vary depending on the vertebrate species in thickness and severity.

The preparation of the submucosal tissue for use according to the invention is in US 4,902,508 described. A vertebrate intestinal segment, preferably of swine, sheep or bovine species, but without excluding other species, becomes abducted by a longitudinal sweep to remove the outer layers comprising the smooth muscle tissue and the innermost layer, ie, the lumenal portion of the Tu nica mucosa, subject. The submucosal tissue is then rinsed with saline and sterilized if necessary.

The Multilayer submucosal tissue graft constructs of the invention can with conventional Sterilization techniques are sterilized, including glutaraldehyde tanning, Formaldehyde tanning at acidic pH, treatment with propylene or Ethylene oxide, gas plasma sterilization, gamma radiation, electron beam, Peracetic acid. Preference is given to sterilization techniques which show the mechanical strength, the structure and biotropic properties of the submucosal tissue not adversely affect. For example, strong gamma radiation at the leaves cause a loss of firmness from submucosal tissue. To preferred sterilization techniques include exposure to peracetic acid the graft, 1-4 Mrad gamma irradiation (more preferably 1-2.5 Mrad gamma irradiation), Treatment with ethylene oxide or gas plasma sterilization; peracetic acid is the most preferred sterilization procedure. typically, The submucosal tissue becomes two or more sterilization processes subjected. After the sterilization of the submucosal tissue, for example Through chemical treatment, it can be made into a plastic envelope or foil and again with electron or gamma rays be sterilized.

The Submucosal tissue may be stored in a hydrated or dehydrated state. Freeze-dried or air-dried submucosal tissue can be moistened again and without significant loss of its biotropic and mechanical Properties used according to the invention become.

at an embodiment of the invention become large-scale yielding leaves formed from submucosal tissue from many strips of submucosal tissue. The dimensions of the individual submucosal tissue strips are not critical and the term "submucosal tissue strip" as used herein is intended to mean submucosal tissue of one or more vertebrate sources or organs in one huge Variety of sizes and shapes include. In one embodiment the strips are formed from a peeled part of intestinal tissue, optionally, but preferably, cut and laid flat becomes elongated Strip submucosal tissue with two generally parallel sides and opposite To form ends. As used herein, the term "submucosal tissue sheet" is intended to include tissue constructs having multiple Submucosal tissue strips, the strips superimposed so overlapping they are a construct with a larger surface than each of the individual for form the formation of this construct used strips. The term "submucosal tissue coating" refers to the individual layers of a multi-layered submucosal tissue construct.

Standardized become large-area submucosal tissue leaves according to the invention by overlapping single submucosal tissue strips and applying pressure to the overlapping ones Parts formed, wherein the tissues are fused together. In one embodiment will put pressure on the overlapping parts under conditions that cause drainage of the submucosal tissue allow. The large-area submucosal tissue leaves can either be formed as Homolaminares or heterola minares sheet. The term "heterolaminar" as used herein refers to a multi-layer fabric with one at different points of the uniform Graft construct of different numbers of submucosal layers, the superimposed (and merged) are. The term "homolaminar" as used herein refers to a multi-layered tissue graft construct with a uniform Number of submucosal layers in all parts of the uniform Graft.

In an embodiment comprises the process of forming large submucosal the steps overlap at least part of a submucosal tissue strip having at least a part of a second submucosal tissue strip and application of pressure at least on the overlapping Part under conditions involving drainage of the submucosal tissue allow. The extent of the overlap between adjacent submucosal tissue strips may vary depending on the intended Use and the desired Properties of the large-area graft changed provided that at least one Part of each submucosal tissue strip with one part of another Submucosal tissue overlaps. The applied pressure fuses the submucosal tissue strips the overlapping one Share with each other and create a compliant uniform heterolaminares Submucosal tissue.

In another embodiment, a uniform homolaminar submucosal tissue sheet can be made from submucosal tissue strips. The method of forming the homolaminary tissue graft construct comprises the steps of forming a first submucosal tissue layer in which submucosal tissue strips are juxtaposed on a first surface. The submucosal tissue strips of the first layer are disposed adjacent to each other so that the edges of the individual strips touch each other without substantially overlapping. The first submucosal layer then becomes a second sub Mukosagewebeschicht launched. The submucosal tissue strips of the second layer, similar to the submucosal tissue strips of the first layer, are disposed adjacent to each other (ie, disposed adjacent each other such that the edges of the individual strips touch without significantly overlapping). In one embodiment, the second layer submucosal tissue strips are aligned in the same direction as the first layer submucosal tissue strips but offset from the first layer submucosal tissue strips such that the contacting edges of the individual submucosal tissue strips of the first layer are bridged by the submucosal tissue strips of the second layer ( please refer 1 ). The overlapping portions of the submucosal tissue strips are then compressed between two surfaces under conditions which permit at least partial drainage of the compressed submucosal tissue, at least one of the surfaces being water permeable.

Advantageous consist of the invention formed both hetero- and homolaminar large-area leaves consisting essentially of submucosal tissue, have increased mechanical strength and a larger surface than any of the formation the submucosa leaves used single strips.

submucosal typically has an inner (lumenal) and an outer (ablumenal) surface. The lumenal surface is the surface facing the lumen of the source organ and is typically adjacent in vivo to an inner mucosal layer, while the ablumenal surface the surface of the submucosa facing away from the lumen of the source organ and typically in vivo in contact with smooth muscle tissue. Several submucosal tissue strips can so overlapped be that the ablumenal surface the lumenal surface, that the lumenal surface the lumenal surface or that the ablumenal surface the ablumenal surface an adjacent submucosal tissue strip contacted. All these overlap combinations of submucosal tissue strips of the same or different Vertebrates or swelling organs produce according to the invention in compression at least the overlapping Parts under conditions which allow drainage of the tissue a large-area submucosal tissue sheet

The submucosal tissue strips of the invention may be conditioned as in US 5,275,826 described to modify the viscoelastic properties of the submucosal tissue. In one embodiment, the submucosal tissue peeled from the tunica muscularis and the lumenal portion of the tunica mucosa is conditioned to have an elongation of not more than 20%. The submucosal tissue is conditioned by stretching, chemical treatment, enzymatic treatment or by exposing it to other environmental influences. In one embodiment, intestinal submucosal tissue strips are conditioned by stretching in the longitudinal or transverse direction to have an elongation of not more than 20%. The conditioned submucosal strips can be used according to the invention for the formation of large-area leaves or multi-layer structures. Alternatively, the submucosa material may be conditioned after the formation of large area sheets or multi-layer constructs to produce a submucosal tissue with an elongation of not more than 20%.

During the Formation of large-area submucosal tissue leaves becomes on the overlapping Parts by squeezing of the submucosal tissue between two surfaces. The both surfaces can made of a variety of materials and in any form be, depending on the desired Form and specification of the uniform graft construct. Typically, the two surfaces are formed as flat plates; you can but other forms include, like sieves, opposing ones Cylinders or rollers and complementary non-planar surfaces. each these surfaces can if necessary heated or perforated. In preferred embodiments At least one of the surfaces is permeable to water. Of the As used herein, water permeable surface includes microporous or macroporous water-absorbent surfaces one. Macroporous Materials include perforated plates or nets made of plastic, Metal, ceramic or wood.

To an embodiment The submucosal tissue is squeezed together by overlapping Submucosal tissue strips on a first surface and a second surface the exposed submucosal surface hangs up. Then a force is applied, which the two surfaces against each other suppressed, wherein the submucosal material is compressed between both surfaces. The pressure force can be determined by any method known to the person skilled in the art be generated, including of performing the device through a pair of nip rolls (the distance the surfaces the rolls smaller than the original one Distance between both plates is), attaching a weight on the top plate and using a hydraulic press or of atmospheric Pressure on the two surfaces.

In a preferred embodiment The submucosal tissue strips are subjected to conditions which a drainage of a submucosal tissue simultaneously with compression. The term "conditions, which a drainage of the submucosal tissue " include any mechanical or environmental condition, which at least at the overlap points promotes the removal of water from the submucosal tissue or initiates. To the drainage of the compressed submucosal tissue, at least one of the two compressing the tissue Surfaces permeable to water. The drainage If necessary, the tissue can be made by applying absorbent material, by heating of the tissue or by blowing air over the exterior of the two compressive surfaces be increased.

The multiple submucosal tissue strips typically contribute 12-48 h Room temperature compressed, although heat can be applied. For example, on the exterior of the compressing surfaces an electric blanket are placed, reducing the temperature of the compressed Tissue to about 40 to about 50 ° C. is increased. The compression time of the overlapping parts is usually through the extent of drainage of the tissue. Application of heat increases the drainage rate and thus diminishes the duration while the overlapping Tissue parts must be compressed. Typically, the tissue is compressed so long that a stiff, but flexible material is created. Sufficient drainage The tissue is also affected by the increase in the impedance of a Tissue flowing electric current displayed. When the impedance is around 100-200 ohms has risen, the tissue was sufficiently dehydrated and the pressure can be lifted.

The Compressed submucosal tissue can be seen from the two surfaces uniform compliant large-area tissue construct be removed. The construct may be further processed (i.e., cut, folded, sewn etc.) in order to adapt to different medical applications, which is the submucosa material according to the invention need, customized to be.

Possibly. can while Apply a vacuum to the submucosal tissue for compression. The applied vacuum is amplified the drainage the tissue and can assist the compression of the tissue. alternative Applying the vacuum can be the sole compression force to press together the overlap Provide parts of the plurality of submucosal tissue strips. For example will be the overlapping Submucosal tissue is laid out between two surfaces, of which preferred a water-permeable is. The device is covered with absorbent material to Absorb water, and with a breather blanket, to an air outlet too enable. The device is then placed in a vacuum chamber and placed in a vacuum, generally between 14 to 70 inches Hg (7-35 psi, 48-241 kPa). Prefers the applied vacuum is about 51 inches Hg (25 psi, 172 kPa). Possibly. can be placed on the top of the chamber an electric blanket to the Submucosal tissue during to heat the compression. For this embodiment suitable chambers are known in the art and include any device provided with a vacuum connection is. The resulting decrease in atmospheric pressure works with the two surfaces in the compression and simultaneous drainage of the submucosal tissue together.

Possibly. can large-area tissue transplants in different forms for the tissue transplantation applications are made. For example can for the Application for organ reconstruction the large leaves in the form of hemispheres or pockets are formed. Such a shaped construct is beneficial in replacing large areas of the bladder or the stomach. These shaped submucosal tissue constructs can be used with usual Techniques like cutting and sewing of the tissue to the desired Be brought form.

alternative can Submucosal tissue strips by a simple manufacturing process to a big one Submucosal tissue sheet be shaped with non-planar shape. The procedure comprises the steps to insert multiple submucosal tissue strips between two complementary ones non-level surfaces and squeezing the overlapping Submucosal tissue between the two surfaces. The complementarily shaped surfaces are shaped so that the both surfaces compressed can be So that the surfaces lie close to each other without an essential between them To leave bubble. Preferably, at least one of the two complementary surfaces is permeable to water.

One method of forming a shaped submucosa construct involves placing a plurality of submucosal tissue strips on a non-planar shaped porous surface such that the submucosal tissue conforms to the contour of the porous surface. Preferably, the submucosal tissue is applied to the porous without stretching the material, however, the submucosal tissue may be stretched to cover to facilitate the shaped porous surface. Each submucosal tissue strip is positioned on the porous surface so as to overlap at least a portion of an adjacent submucosal tissue strip. The overlapping submucosal tissues are covered with a second surface complementarily shaped to the first porous surface and pressure is applied to compress the submucosal tissue between the two surfaces under conditions which permit dewatering of the submucosal tissue.

alternative can the large-sized leaves according to the invention by a molding process be formed in non-planar shape, the submucosal tissue from a porous one Stamp under draining Conditions in non-planar shape is pressed, so that the molded Tissue graft maintains its shape. It is preferred in one Such a method is a large-area multilayer sheet used.

multilayer Submucosal tissue constructs according to the invention by overlapping a part of a Submucosal tissue strip with a portion of another submucosal tissue pathway educated. On similar Way you can According to the invention, large-area multilayer tissue transplant constructs by overlapping a submucosal tissue sheet (formed as described above) with formed at least part of a second submucosal tissue sheet become. Size and physical Properties of the multilayer tissue graft construct may be demonstrated by the number of overlapping ones Submucosal tissue strips and the percentage of overlapping Part of each strip to be controlled.

According to the invention the multi-layered tissue graft constructs by overlapping a part of a submucosal tissue strip with a part of a another submucosal tissue strip to form a first leaf educated. On the overlapping Parts of the first leaf continue to become submucosal tissue strips Formed a second sheet, the edges of the Strip of the second sheet possibly an acute angle with the strip edges of first sheet can form and wherein the second sheet thus formed is coplanar with the first one Leaf is. The submucosal tissue strips of the second leaf can do so be positioned that at least a part of a Submukosagewebestreifens the second sheet with at least overlaps a part of another submucosal tissue strip of the second leaf. To provide further submucosal tissue layers, others may be provided Submucosal tissue strips on the overlapping Parts of the first and second sheets are launched. The several Submucosal tissue layers are then compressed under dehydrating conditions and form a multi-layered heterolaminar submucosal tissue construct with a surface bigger everyone the for the formation of the multi-layer construct used single submucosal tissue strips.

In an embodiment of the invention Submucosal tissue is cut into strips, each strip all have mutually parallel sides, and to form heterolaminar inventive Multilayer sheets used. In this embodiment The submucosal tissue strips of the second layer become overlapping Parts of the first layer placed so that the edges of the Submukosastreifen the first layer at an angle to the edges of the submucosal strips the second layer. The overlapping Submucosal tissue is compressed under dehydrating conditions and form the heterolaminar multilayer sheet. These leaves can be cut will not sprout without delamination and delamination when soaking in Water over a period of time (more than an hour), which is the time to implant of the leaf needed in a host Time equals. If the leaf is implanted correctly, so that it at all Sewn pages is, should no delamination occur after implantation.

According to the invention, a tissue graft construct having pseudoisotropic properties is formed from submucosal tissue strips. The pseudo-isotropic tissue graft constructs comprise a plurality of submucosal tissue strips fused together in a multilayer structure without adhesives or sutures. The pseudo-isotropic multi-layer tissue graft is made according to the invention from at least three intestinal submucosal tissue strips peeled off both the tunica muscularis and the lumenal portion of the tunica mucosa of a warm-blooded vertebrate. Each of the intestinal submucosal tissue strips is characterized by a longitudinal axis corresponding to the predominant orientation of the collagen fibers in the submucosal tissue strips. The method of producing the pseudoisotropic graft constructs comprises placing a first submucosal tissue strip on a first surface, placing at least two further submucosal tissue strips on the first strip such that the longitudinal axis of each individual submucosal tissue strip makes an angle of approximately 180 ° / N with the longitudinal axes of at least two forms other submucosal tissue strips forming the terolaminar graft, where N is the total number of submucosal tissue strips (see 2 ). For example, a pseudoisotropic graft construct formed from four (4) submucosal tissue strips has an angle of 45 ° (180 ° / 4 = 45 °) between the central longitudinal axes of each strip and two of the three others form the graft construct denden strips (see 2 ). The submucosal tissue (at least the overlapping portions) is then compressed between the first and second surfaces. In one embodiment, the tissue is compressed under conditions that permit at least partial drainage of the submucosal tissue, and in a preferred embodiment, at least one of the surfaces is water permeable.

Large area tissue graft constructs with pseudoisotropic properties can also be made from large area submucosal tissue sheets. These pseudoisotropic tissue graft constructs comprise multiple layers of large area submucosal tissue sheets containing overlapping submucosal tissue strips. The submucosal tissue sheets containing overlapping submucosal tissue strips may be formed as homolaminar or heterolaminar tissue constructs as described in this specification above (see 3 ).

One Manufacturing process for a large area multi-layer tissue graft construct having pseudoisotropic properties includes forming a first one Submucosal tissue sheets from multiple submucosal tissue strips and overlaying of the first leaf with at least two other leaves. The have individual submucosal tissue strips contained in each leaf a longitudinal axis, which of the predominant orientation of the collagen fibers in the Submucosal tissue strip corresponds. The first sheet is on a first surface formed in which the individual submucosal tissue strips so overlaps be that every strip is aligned with the adjacent strips and the longitudinal axis of all submucosal tissue strips are parallel to each other. Therefore, the collagen fibers of the first Leaf predominantly oriented in one direction, leaving the leaf a longitudinal axis according to the predominant orientation of the collagen fibers can be attributed.

As described above large-area submucosal tissue sheets of overlapping Submucosal tissue are formed, where they are either heterolaminare or homolaminar submucosal tissue sheets. Both heterolaminar as well as homolaminar leaves are to form the large area pseudoisotropic tissue graft constructs of the present invention suitable.

To Placing the first submucosal tissue sheet on the first surface on the first leaf further layers of submucosa leaves on the same way as the first leaf is formed (i.e., each submucosal tissue sheet of the multilaminate contains overlapping Submucosal tissue strips, with the longitudinal axes of each leaf contained submucosal tissue strips are substantially parallel to each other). Each individual sheet is placed on another sheet, so that the longitudinal axes the submucosal tissue strip of the laid-up sheet an angle from about 180 ° / s (S = total number of submucosal tissue sheets) with the longitudinal axes the submucosal tissue strip of at least two of the others Form multi-layer construct forming sheets. After hanging up of all leaves become the submucosal tissue leaves between a first and a second surface under conditions which an at least partial drainage of the Allow compressed submucosal tissue compressed. In a preferred embodiment At least one of these surfaces is water permeable.

In an embodiment after several submucosal tissue strips overlap were overlapping Parts for removing trapped air and larger amounts of water treated before the strips fused into a single sheet become. Generally, air bubbles and larger amounts of water are applied one over the surface the overlapping Squeezed out parts of moving compression force. The compression force can take the form of an over the surface the overlapping Parts may have rolled cylinders or, alternatively, the overlapping ones Parts are passed between two or more rollers, the distance between the surfaces of opposing rolls being smaller than the thickness of the submucosal sheet. The overlapping parts can then, if necessary, dehydrating for another time Conditions are compressed to the multiple strips according to the invention to a single submucosal tissue sheet to merge.

The excess parts pseudoisotropic multilayer grafts (i.e., those parts of the graft with a number of layers smaller than N or S) can removed after formation of the multilaminate. Furthermore, the mechanical properties of the multi-layer submucosa material needs the medical application by the overlap percentage set between adjacent submucosal tissue strips, the angle adjacent layers to each other changed, the water permeability and / or the composition of the compressive surfaces changes the shape the compression surfaces selected and for the compression of the overlapping Submucosal tissue applied force is changed.

The pseudo-isotropic according to the invention Submucosal material can be further modified so that it has a Contains variety of perforations. Recent experiments have shown that the remodeling process is implanted at Multilayer submucosal tissue graft constructs slower than in submucosal tissue grafts with one or two layers is. Furthermore Multilayer submucosal tissue graft constructs tend to in soft tissue areas (such as the muscular body walls of rats) during the first 14 to 28 days after implantation, tissue fluid in cyst-like pockets to accumulate between adjacent layers. Fluid pockets are the Wound healing, because they delay the ingrowth of connective tissue, a for bacteria conducive Environment and the attachment of natural (own) body tissue, promotes healing and tensile strength, prevent.

It it was found that Perforating the multilayered graft constructs the remodeling properties of the graft in vivo and the adhesion the tissue graft layers improved together. It is believed, that the Perforations the contact of the submucosal tissue with endogenous fluids and promotes cells (by increase of the surface of the implanted graft) and that the perforations also as Serve to conduct, causing the extracellular fluid through the graft can pass through.

Designated according to the invention the term "perforation" a hole that is itself extends through the whole graft construct. However, tissue transplant constructs are present with "holes", here as a cavity defined, which penetrates into the tissue, but not by the whole graft extends, also in the area of the present Invention. Distance and size of the perforations as well as their depth of penetration into the tissue can vary depending on the desired mechanical strength, porosity, Size and thickness (Number of layers) and others on the medical application the tissue transplant related factors are changed. The size of the perforations ranges from 0.5 to 3 mm, more preferably from 0.6 to 2 mm. In a embodiment the perforations are equidistant at a distance of 2 to 20, more preferably from 3 to 7 mm.

typically, the perforations are formed in the submucosal tissue while the Tissue remains at least partially hydrated. In the inventive pseudoisotropic Multilayer submucosal tissue materials become the perforations preferably prepared after formation of the multilayer construct, if the fabric to a water content of about 10-20% by weight (10-20% hydrated) was dried. Sufficient drying of the tissue can be achieved by Weigh the fresh tissue and dry to 10-20% of the weight Fresh weight or by impedance measurement as described above become. After perforation, the submucosal tissue becomes a final sterilization subjected and stored as described above.

In an embodiment can holes (which only partially extend through the tissue) or perforations be formed on both sides of the tissue graft. In addition, can the fabric can be modified to include both perforations also holes, which only partially extend through the tissue contains. Further The submucosal tissue can be modified to have a Variety of holes having different subgroups of holes compared to other holes extend into different depths into the tissue. This can be, for example can be achieved by coating the individual submucosal tissue before overlapping perforated to form the multilayer construct. If some of the Layers are not perforated or the perforations of the individual Layers do not align, have formed multilayer constructs holes, which extend to different depths in the tissue. Prefers the tissue is perforated with uniform distribution across the tissue graft surface, causing a series of holes is formed, which is a liquid Connection between a first planar surface to a second opposite even surface of the graft.

In an embodiment The perforations are perpendicular to the surface of the tissue graft construct trained, d. H. the longitudinal axis The perforation / hole forms with the surface of the graft defining plane an angle of 90 °. Alternatively, the Perforations are formed so that the axis of the perforation not perpendicular to the surface of the graft (i.e., so that the perforation / Hole delimiting wall parallel longitudinal axis at an angle different from 90 ° forms with the plane of the graft). According to one embodiment are the perforations with an angle of 45 to 90 ° with respect to the graft surface educated.

Multilayer tissue grafts can be cut without spitting and do not delaminate when soaked in water for a period of time (more than one hour) equal to the time required to implant the leaflet into a host. However, multi-layered submucosal tissue transplants tend to be In the soft tissues (such as rat muscular body walls), tat constructs tissue fluid in cyst-like pockets between adjacent layers during the first 14 to 28 days after implantation. Perforations of the pseudoisotropic multi-layered graft constructs of the invention reduce the accumulation of fluid between the layers of the multi-layered construct by providing a drain through which fluid can flow out of the tissue. In addition, the perforations have a "staple" effect which enhances the adhesion of the layers to one another.

• 1. Erhöhter Durchtritt von Flüssigkeiten (einschließlich von Gewebeflüssigkeit) durch das Material und
• 2. Erhöhte Adhäsionskraft zwischen benachbarten Schichten.

Accordingly, the provision of holes in multilayer tissue grafts over all or part of the thickness over non-perforated multilayer sheets offers the following advantages:

• 1. Increased passage of fluids (including tissue fluid) through the material and
• 2. Increased adhesion between adjacent layers.

The Submucosal tissue may be known by a variety of those skilled in the art Devices are perforated. The one used for perforating Procedure is not critical, provided that the composite structural integrity of the submucosal tissue.

In preferred embodiments leads the Perforation of the submucosal tissue not essential to the removal Proportions of the tissue. For example, the perforations are through Pressing a pointed solid object through the tissue formed while being of introduction of the solid object, the tissue is pushed aside as opposed to Drilling out the material. Other means of perforating the tissue include the use of shooting, cutters, laser beams or enzymatic / chemical treatment.

In an embodiment The submucosal tissue is made by pressing a pin or a solid needle perforated in / through the tissue. Typically, the formation the perforation a fixed needle of 20-23 gauge (0.51-0.58 mm) used. This will help in the formation of perforations no significant amount of tissue removed, but it becomes a part each layer torn and pushed into the adjacent layer, causing a staple effect arises. This "staple" effect can be further enhanced, if you have some of the perforations from one side of the transplan tat and the remaining perforations from the opposite side generated the graft.

4 Figure 1 illustrates an embodiment of a device for perforating the submucosal tissue graft constructs. The device comprises a base plate ( 1 ) and a plurality in the base plate ( 1 ) embedded stainless steel needles ( 2 ) extending through the surface of the base plate to the outside. The base plate comprises parts of epoxy (E) and Delrin (D) and has a length of 3.2 inches (81 mm), a width of 1.85 inches (47 mm) and is 0.5 inches (12.7 mm) thick. According to this embodiment, the needles ( 2 ) are substantially parallel to one another and form an angle of 90 ° with the surface of the base plate. The needles ( 2 ) have a diameter of 0.040 inches (0.10 mm) and a mutual distance of 0.264 inches (6.7 mm) (center to center of adjacent needles) at a distance of 0.4 inches (10.2 mm) from the edge of the Device and stand out 0.25 inches (6.35 mm) from the base plate. Thus, the device contains a total of 50 needles.

example 1

From vertebrate intestinal tissue was after the in the US 4,902,508 described method submucosal tissue produced. The submucosal tissue strips were prepared from a section of intestinal tissue of a warm-blooded vertebrate, the section comprising the tunica submucosa, peeled off from both the tunica muscularis and at least the lumenal portion of the tunica mucosa, of that section of intestinal tissue. The intestinal tissue section was cut along the longitudinal axis of the section and laid flat. The fabric was then further cut into a series of strips with generally parallel sides.

Several Submucosal tissue strips were placed on a perforated stainless steel plate arranged by 12 times 12 inches (30.5 by 30.5 cm), with one part of a submucosal tissue strip overlapped a portion of the adjacent submucosal tissue strip. Then a second perforated stainless steel plate of 12 by 12 inches was placed the submucosal tissue is placed. The stainless steel plates used in this embodiment had perforations of 0.045 inches (1.14 mm), straight centered (straight Center) with a distance of 0.066 inches (1.68 mm) from each other. A weight of 50-100 pounds (22.7-45.3 kg) was placed on the second stainless steel plate and the tissue was compressed at room temperature for 24 h.

Example 2

submucosal tissue were prepared as described in Example 1. Several submucosal tissue strips were laid between two perforated stainless steel plates, so the existence Part of a submucosal tissue strip overlapped part of the adjacent submucosal tissue strip. The device from "plate submucosa plate" was placed on a flat surface laid and with an absorbent Covered material to drain off the water, and with a venting foil, to an air outlet too enable. The device was then sealed in a nylon bag containing had a vacuum connection. A vacuum (greater than 28 inches Hg - 950 hPa) was applied to suck the air from the vacuum bag and the resulting air pressure drop compressed and dehydrated the Submucosal tissue at the same time. After 24 h applying the vacuum was the sheet produced moist and very flexible. There were no seams of Submucosal layers visible and those determined by the bullet burst test Strength of a prototype sheet of 8 thicknesses was 80 pounds (36.3 kg).

Example 3

submucosal tissue were prepared as described in Example 1. The submucosal tissue strips were placed on a sieve so that one of a submucosal tissue strip a portion of an adjacent submucosal tissue strip overlapped. After the sieve was covered with a layer of submucosal tissue, was a second layer of submucosal tissue on the first layer so that the Edges of the Submucosal strips of the second layer make an angle with the edges of the Submucosa stripes of the first layer formed.

After this all submucosal tissue strips were placed on the sieve place another sieve on the submucosal layers and the "sieve submucosal tissue sieve" sandwich was compressed with a weight and dried. The procedure yielded a dried large area submucosa leaf, which was peeled off the sieve as a unitary graft construct.

Example 4

sterilization of submucosal tissue with peracetic acid

submucosal is in a peracetic acid / ethanol solution for 2 h at room temperature at a ratio of 20: 1 or more (ml Peracetic acid: grams Submucosal tissue) soaked. The peracetic acid / ethanol solution contains 4% ethanol, 0.1% (v / v) peracetic acid and the rest is water. The 1-percent. Peracetic acid component is a dilution of a 35-proz. Peracetic acid stock solution, the is commercially available and defined in Table 1. The submucosal tissue is preferred while soaking in the peracetic acid solution a shaker shaken. After 2 h, the peracetic acid solution is poured off and by the equivalent Amount of Ringer's lactate solution or phosphate buffered saline (PBS) replaced and (with shaking) Soaked for 15 minutes. The submucosal tissue will be four more wash cycles with Ringer's lactate solution or PBS and then washed with sterile water for a further 15 min.

Table 1

Sterilization of submucosal tissue with ethylene oxide

To Production of multilayer constructs under sterile conditions the material is packaged and a second sterilization cycle (Final sterilization) subjected. The fabric can be packed in plastic, the for ethylene oxide permeable is and sterilized by ethylene oxide with the method Are known in the art. In essence, the packaged material the ethylene oxide for 4 hours at 115 ° F (46 ° C) exposed. While sterilization, the material is at least 75 min of 4 h below Maintained at 65% relative humidity. The high humidity intensifies the Absorption of the ethylene oxide through the tissue. After 4 hours, ethylene oxide, Ethylene chlorohydrin and ethylene glycol rinsed with nitrogen and air.

Example 5

Bump burst test by means of compression cage and MTS tensile tester

The Strength of multi-layer submucosal tissue grafts is under Application of the tensile tester a material testing system (MTS) determined. The multi-layer tissue construct is fastened in a clamp with four-sided frame (sample clamp), about the tension over distribute the tissue construct evenly. The mounting height at the beginning is adjusted so that the top of the ball is immediately located below the sample level. The handle of the sample clamp becomes in his highest Position raised so that the jaws of the clamp can take the sample. The Submucosal tissue construct is cut to match the sample clamp. The opening the clamp has a diameter of one-quarter inch (44.5 mm). At the periphery of the sample, an excess of material of one-half inch (12.7 inches) should be allowed mm), so that enough clamping surface is secured. The submucosal tissue is placed in the jaws of the brace inserted and fixed, the clamping force by an adjustment screw is regulated at the upper cheek.

The trapped submucosal tissue is then passed over it at a controlled rate Metal ball pushed down, being for regulating and measuring the force acting on the sample Interface with train testing software is used. The force is increased until the sample breaks. Break means the maximum load, the first appearance of the sphere by visible unnatural discontinuities in the sample plane equivalent. If the highest Situation is reached before the break, the software intervenes and ends the try. The peak load displayed on the Microprofiler 458.01 is recorded and the sample is removed.

Example 6

A multilayer tissue graft construct was prepared as follows:
From vertebrate intestine a plentiful amount of submucosal tissue is prepared, cut, laid flat and disinfected with peracetic acid as described in Example 4 (for a 10 x 15 cm device, about 70 g of submucosal tissue is needed). After sterilizing the fabric with peracetic acid, rubber gloves, face mask and hair protector should be worn to minimize contamination with organic material and dust particles.

submucosal tissue be on a first stainless steel plate with the desired Orientation hung up. The used stainless steel plates are with round perforations of 0.045 inches (1.14 mm) with straight aligned Punched straight centers that are 0.066 inches (1.68 mm) away from each other. After formation of a submucosal layer the submucosal tissue is smoothed to remove air bubbles. Additional layers are applied until the device is ready. With scissors becomes excess material removed around the multilayer structure. The weight of the submucosa multilaminate is noted. On the multilayer construct is a second stainless steel plate (perforated with 0.045 inches (1.14 mm) round perforations on straight Center points, 0.066 inches (1.68 mm) apart.

The Multilayer construct may optionally be "roll-crushed" to contain trapped To remove air and water. For squeezing the material Both are the perforated metal plates surrounding the submucosal tissue between two polypropylene sheets (Kimberley Clark, class 100 "crew Wipe ") and the whole arrangement is between two layers of nylon wrapper (ZipVac, Auburn WA) placed larger than 1 '× 1' (305 × 305 mm) are. Then a loaded cylinder several times (at least three times) over the Arrangement rolled.

Around to perforate the multi-layered submucosal tissue graft construct, the arrangement is partially disassembled to the upper surface of the Tissue graft, and immediately to the top Submucosal tissue layer is placed on a piece of nylon wrapping film. The multi-layered submucosal tissue graft construct then becomes vice versa on a work surface made of polystyrene foam (styrofoam) and the first stainless steel plate is removed carefully. The exposed submucosal surface becomes then with a piece Covered nylon wrapper. Then the tissue graft construct becomes perforated and then removed the upper nylon wrapper. The multi-layered submucosal tissue graft construct then becomes Turned over again and returned to the perforated stainless steel plate. The nylon wrapper is removed from the upper submucosal surface and a second perforated stainless steel plate on top of the multilayered submucosal tissue graft construct.

The multi-layered submucosal tissue graft construct is then compressed under dehydrating conditions as follows:
A layer of absorbent material (NuGauze), larger than the perforated plates, is placed on a table top (or other smooth surface). The stainless steel plates with the multi-layered submucosal tissue graft construct therebetween are placed on the absorbent material. Another layer of absorbent material (about the same size as the first layer) is placed on the stainless steel plates. A deaerating film (ZipVac, Auburn, WA) is placed on the absorbent material. Preferably, the venting foil is slightly larger than the objects covered by it.

Possibly. can Electrodes are placed in contact with the submucosal tissue, to measure the impedance across the tissue. Typically that will Tissue compressed until a stiff but flexible material is produced. Sufficient drainage of the material is due to an increase in the impedance for that Tissue flowing through electric current displayed. When the impedance is around 100-200 ohms has risen, the tissue was sufficiently dehydrated and the pressure can be lifted.

Around the device and the area to be compressed by vacuum, a double-adhesive tape surround is placed on the table top. The protective film is peeled from the belt and a piece of nylon wrapping film, already provided with a nozzle port, is placed on the area covered by the tape (see 3a and 3b ) and fixed on the tape. The heating blanket, if used, and the vacuum pump are switched on. The bag is examined for wrinkles (if necessary smooth) and for insufficient sealing between the adhesive tape and the nylon packaging film (correct if necessary). The vacuum is drawn to a vacuum of 25-30 psi _vac (172-207 kPa). After vacuum treatment to the desired level of moisture (about 24 hours), the pouch seal on the adhesive tape is opened, the vacuum pump is turned off, and the uniform perforated multi-layer submucosal tissue allograft construct is removed. Parts of the fabric transplant that have not received the full amount of overlap can be trimmed with scissors.

Example 7

The Submucosal tissue graft construct may also be present after formation of the uniform multilayer construct can be perforated as follows. The multilayer construct is formed according to Example 3. The sandwich of sieve and submucosal tissue was removed from the dryer removed and the tissue was perforated. The graft was perforated by inserting at several points of the graft surface Nail was inserted between the meshes of the mesh and pressed through the tissue. The perforated multi-layer submucosal tissue then became square cut (4½ x 4½ inches; 114 × 114 mm) and marked for identification.

Example 8

A perforated pseudoisotropic laminate construct was prepared as follows: Submucosal tissue strips were placed on a wire screen in 4 layers. The first layer was deposited directly on the screen and the remaining three layers were laid on it at angles of 45, 90 and 135 degrees relative to the first layer (see 2 ). A second sieve was placed on the submucosal tissue and the tissue was placed between the sieves and clamped to the drying rack. A fan was set up in front of the rack and switched on. Using the screen as a guide, holes in the checkerboard pattern were punched through the fabric (ie, the perforated areas were alternately used). Accordingly, the pattern appeared as follows:

The Perforation of the tissue was stopped before completion because the tissue of the submucosal tissue tore. Therefore, the submucosal tissue was kept at high setting for 25 minutes Fan dried. Then the rest of the perforations according to the original one Pattern attached.

you let that go Sheet over Dry the night, took it out, cut it into squares and tagged it it for identification.

Claims (44)

Tissue graft construct comprising a compliant Layer of submucosal tissue, which consists of a variety of overlapping Strip is formed from submucosal tissue, with the yielding layer from submucosal tissue a larger surface area than each of the individual to form the compliant layer strips used in submucosal tissue. The tissue graft construct of claim 1, wherein the strips of submucosal tissue essentially from the tunica submucosa, the muscularis mucosa and the stratum compactum of the tunica Mucosa exist. The tissue graft construct of claim 1, wherein the strips are conditioned from submucosal intestinal tissue, a Elongation of not more than 20%. A method of forming a unitary homolaminary tissue graft construct from a plurality of strips of submucosal tissue peeled from both the tunica muscularis and the luminal portion of the tunica mucosa of a warm-blooded vertebrate, which construct has a greater surface area than any of the individual used to construct the construct Strip, the method comprising the steps of: forming a first layer of submucosal tissue by placing strips of submucosal tissue on a first surface, the strips of submucosal tissue of the first layer adjacent to each other and without substantial overlap with each other; Overlapping the first submucosal tissue layer with a second layer of submucosal tissue strips, wherein the second layer submucosal tissue strips are adjacent to and in contact with each other without substantial overlap, and wherein the submucosal tissue strips are opposite the second layer the first layer submucosal strips are offset so that at least a portion of the contacting edges of the individual submucosal tissue strips of the first layer are bridged by strips of submucosal tissue of the second layer; and compressing the overlapping portions of the strips of submucosal tissue between the first surface and a second surface under conditions that permit at least partial dehydration of the compressed submucosal tissue. The method of claim 4, further comprising Step of applying a vacuum to the submucosal tissue during the Compression of the same includes. The method of claim 5, wherein the applied vacuum with the two surfaces cooperates to compress the submucosal tissue. The method of claim 4, further comprising Step of applying heat on the submucosal tissue during the compression of the same. The method of claim 4, wherein at least one the upper surfaces permeable to water is. The method of claim 4, wherein the two surfaces are not complementary planar surfaces are, with a compression between the two surfaces Tissue graft construct generated, which is brought into a mold is to coincide with these surfaces. Multi-layered tissue graft construct, which consists essentially of submucosal intestinal tissue, wherein The multi-laminated construct is shaped to be pseudoisotropic To exhibit properties. The tissue graft construct of claim 10, wherein the submucosal intestinal tissue essentially from the tunica submucosa, the muscularis mucosa and the stratum compactum of the tunica mucosa. The tissue graft construct of claim 10, wherein The submucosal tissue is conditioned to not stretch more than 20%. Process for producing a multi-laminated Tissue graft construct having pseudoisotropic properties, wherein the construct consists of at least three strips of submucosal Intestinal tissue is formed by both the tunica muscularis and the luminal region of the tunica mucosa of a warm-blooded vertebrate peeled wherein each strip of submucosal intestinal tissue is a longitudinal axis has, with the predominant orientation of collagen fibers in the submucosal tissue strips, the method the following steps: Superimposing the first Strip with at least two additional strips of submucosal Tissue, so that the longitudinal axis from each of the individual strips of submucosal tissue an angle of about 180 ° / N with the longitudinal axes at least two other strips of the superimposed submucosal tissue where N = the total number of strips of submucosal Tissue is; press together of at least the overlapping one Areas of strips of submucosal tissue between two surfaces below Conditions that involve at least partial dehydration of the compressed submucosal Enable tissue. The method of claim 13, wherein at least one the surfaces permeable to water is. A method of making a multi-layered tissue graft construct having pseudo-isotropic properties, wherein the construct is formed from a plurality of sheets of submucosal intestinal tissue, each of the sheets having overlapping strips of submucosal tissue extending from both the tunica muscularis and the luminal portions of the tunica mucosa warm-blooded vertebrate, each of the strips having a longitudinal axis corresponding to the predominant orientation of the collagen fibers in the submucosal tissue strips, the longitudinal axes of the submucosal tissue strips of each layer being substantially parallel to each other; A method comprising the steps of superimposing the first layer of submucosal tissue with at least two additional layers of submucosal tissue so that the longitudinal axes of the strips of submucosal tissue from a layer are at an angle of approximately 180 ° / S with the longitudinal axes of the strips of submucosal tissue of at least two of the superimposed layers of submucosal tissue, where S = the total number of layers of submucosal tissue; and compressing the submucosal tissue sheets between two surfaces under conditions which permit at least partial dehydration of the compressed submucosal tissue. The method of claim 15, further comprising the step of applying a vacuum to the submucosal tissue while the compression of the same. The method of claim 16, wherein the applied Vacuum with the two surfaces cooperates to compress the submucosal tissue. The method of claim 15, further comprising the step of applying heat on the submucosal tissue during the compression of the same. The method of claim 15, wherein at least one the surfaces permeable to water is. The method of claim 15, wherein the two surfaces are not complementary Planar surfaces are, the compression between the two surfaces constructing a tissue graft which is shaped to conform to these surfaces. Multi-layered tissue graft construct with pseudoisotropic properties, the construct consisting of a Variety of layers of submucosal tissue is formed, each one the overlapping layers Strip of submucosal tissue, each of the strips a longitudinal axis has, with the predominant orientation of collagen fibers in the submucosal tissue strips korres pondiert, wherein the longitudinal axes the strip of submucosal tissue of each of the respective layers are substantially parallel to each other. Multi-layered tissue graft construct according to claim 21, wherein the overlapping ones Strips of submucosal tissue have submucosal tissue strips, each of which has an area coincident with an area of a adjacent strip of submucosal tissue is overlapped. Multi-layered tissue graft construct according to claim 21, wherein the overlapping ones Strip of submucosal tissue a first layer of submucosal Tissue strips having a second layer of submucosal Layered fabric strips is where the strips of submucosal tissue from each of the respective Layers adjacent to and in contact with each other without substantial overlap placed between the adjacent strips of submucosal tissue with the strips of submucosal tissue of the second layer offset with respect to the submucosal strips of the first layer are so that at least a portion of the contact edges of the individual Strip of submucosal tissue of the first layer through the strips are bridged from submucosal tissue of the second layer. The graft of claim 21, wherein the strips from submucosal tissue essentially from the tunica submucosa, the muscularis mucosa and the stratum compactum of the tunica mucosa consist. The graft of claim 21, wherein the strips from submucosal intestinal tissue are conditioned to stretch of not more than 20%. The tissue graft material of claim 21, wherein the submucosal tissue is the tunica submucosa and basilar tissue the tunica mucosa has. The tissue graft construct of claim 1, wherein the submucosal tissue is submucosal intestinal tissue. The tissue graft construct of claim 1, wherein the submucosal tissue of both the tunica muscularis and the luminal area of the tunica mucosa of a warm-blooded vertebrate is peeled off. The tissue graft construct of claim 1, wherein the construct is chemically treated. The tissue graft construct of claim 1, wherein the construct is heated is. The tissue graft construct of claim 29, wherein the construct is heated is. The tissue graft construct of claim 29, wherein treated the construct with a chemical sterilant is. The tissue graft construct of claim 29, wherein the chemically treated construct has an elongation of less than 20% having. A tissue graft construct according to claim 1, which for one Hernia repair, skin grafting, meningeal covering, Gastroschisis recovery or organ tissue replacement is configured. The tissue graft construct of claim 34, which configured for hernia recovery. Using a variety of strips of submucosal Tissue with overlapping Edges in the manufacture of a unitary multi-laminated compliant Layer of submucosal tissue for hernia repair. The method of claim 4, further comprising comprises the step of chemically treating the construct. The method of claim 37, further comprising the step of warming of the construct. The method of claim 4, wherein the construct with a chemical sterilant is treated. The method of claim 37, wherein the chemically treated Construct has an elongation of less than 20%. The tissue graft construct of claim 10, wherein the submucosal tissue is submucosal intestinal tissue. The tissue graft construct of claim 10, wherein the submucosal tissue of both the tunica muscularis and the luminal area of the tunica mucosa of a warm-blooded vertebrate is peeled off. The tissue graft construct of claim 21, wherein the submucosal tissue is submucosal intestinal tissue. The tissue graft construct of claim 21, wherein the submucosal tissue of both the tunica muscularis and the luminal area of the tunica mucosa of a warm-blooded vertebrate is peeled off.